Coordination to lanthanide ions distorts binding site conformation in calmodulin.

Abstract

The Ca²⁺-sensing protein calmodulin (CaM) is a popular model of biological ion binding since it is both experimentally tractable and essential to survival in all eukaryotic cells. CaM modulates hundreds of target proteins and is sensitive to complex patterns of Ca²⁺ exposure, indicating that it functions as a sophisticated dynamic transducer rather than a simple on/off switch. Many details of this transduction function are not well understood. Fourier transform infrared (FTIR) spectroscopy, ultrafast 2D infrared (2D IR) spectroscopy, and electronic structure calculations were used to probe interactions between bound metal ions (Ca²⁺ and several trivalent lanthanide ions) and the carboxylate groups in CaM's EF-hand ion-coordinating sites. Since Tb³⁺ is commonly used as a luminescent Ca²⁺ analog in studies of protein-ion binding, it is important to characterize distinctions between the coordination of Ca²⁺ and the lanthanides in CaM. Although functional assays indicate that Tb³⁺ fully activates many Ca²⁺-dependent proteins, our FTIR spectra indicate that Tb³⁺, La³⁺, and Lu³⁺ disrupt the bidentate coordination geometry characteristic of the CaM binding sites' strongly conserved position 12 glutamate residue. The 2D IR spectra indicate that, relative to the Ca²⁺-bound form, lanthanide-bound CaM exhibits greater conformational flexibility and larger structural fluctuations within its binding sites. Time-dependent 2D IR lineshapes indicate that binding sites in Ca²⁺-CaM occupy well-defined configurations, whereas binding sites in lanthanide-bound-CaM are more disordered. Overall, the results show that binding to lanthanide ions significantly alters the conformation and dynamics of CaM's binding sites.